(1) Field of the Invention
This invention generally relates to virology, and more particularly to the provision of a mammalian cell line that has been genetically engineered to permit the detection and quantitation of the presence of an RNA virus in a biological specimen and a method for detecting RNA viruses using these cells.
(2) Description of Related Art
The methods by which biologically active or infectious viruses are detected at the clinical level have changed little over recent decades. The standard diagnostic assay for viral infections involves inoculation of specimens onto tissue culture cells followed by detection of infectious virus by microscopic observation of a characteristic cytopathic effect. This method has been supplemented by automated methods that detect viral antigen or viral nucleic acid, but an automated method for the detection of infectious virus is not presently available. The automated assays that detect viral antigen often provide the advantages of rapidity and specificity, but they also often lack the requisite sensitivity necessary for a clinically reliable assay. Automated assays that detect the presence of viral nucleic acid have also recently been developed, but such assays detect viral nucleic acid and not infectious virus. The detection of infectious virus is often preferred because it definitively indicates the existence of an ongoing viral infection with active viral replication. Moreover, assays detecting only viral nucleic acid may only be indicative of the presence of a remnant of a past infection or the presence of a latent infection and the treatment necessary for an ongoing infection may be different than that for a latent or past infection. Thus, the provision of a rapid, specific, sensitive and cost-efficient assay for the detection of infectious virus would be a valuable addition to a clinical diagnostic laboratory and to research laboratories needing a rapid and sensitive assay to determine the presence or absence of an RNA virus in a fluid.
Recently, methods for detecting infectious DNA viruses and RNA viruses that replicate through a DNA intermediate, such as HSV and HIV, have been disclosed that utilize a genetically engineered cell line containing a chimeric gene having a reporter gene under the control of a regulatory region that is activated in the presence of active virus to cause expression of the reporter gene product. Rocancourt, et al. J. Virol. (1990) 64:2660-2668; Kimpton, J. and Emerman, M., J. Virol. (1992) 66:4:2232-2239. This approach has proved useful for the detection of DNA viruses and RNA viruses that replicate through DNA, but is not applicable to the detection of RNA viruses that replicate through an RNA intermediate and not through DNA. A primary reason for this is that RNA viruses replicate in the cytoplasm and have no known mechanism which would permit the RNA virus to transactivate a DNA promoter contained within the nucleus of a cell.
Numerous RNA viruses are pathogenic to humans and their diagnosis is important clinically and for various research purposes. The Togavirus family of RNA viruses includes the genus Alphavirus which includes many important viral species such as Sindbis virus, Semliki Forest virus, and pathogenic members such as the Venezuelan, Eastern and Western equine encephalitis virus. Another pathogenic Togavirus is the rubella virus, a virus closely related to the alphaviruses and the causative agent for German measles. Coronaviruses (one of the major causative agents for common colds), and astroviruses (associated with pediatric diarrhea), are also pathogenic RNA viruses. All of these viruses are characterized by a life cycle that include the synthesis of subgenomic RNAs. For example, the Sindbis virus genome consists of a single molecule of single stranded RNA. The genomic RNA is infectious and serves as mRNA and is, by convention, of plus (+) polarity. The 5' two-thirds of the genomic RNA is translated to produce a polyprotein that is processed by co-translational and post-translational cleavage into four nonstructural proteins presumably required for RNA replication. A full-length minus (-) strand complementary to the genomic RNA is then synthesized. This minus strand serves as a template for the synthesis of a new genomic RNA plus (+) strand molecule and as a template for transcription of a subgenomic mRNA molecule. Transcription from the minus (-) strand begins at an internal site to produce the subgenomic mRNA. This internal site is referred to as the junction region or subgenomic RNA promoter region. This region of the Sindbis virus is described in U.S. Pat. No. 5,217,879 issued on Jun. 8, 1993 and is commonly assigned to the assignee of this application. The entirety of U.S. Pat. No. 5,217,879 is herein incorporated by reference hereto. Translation of the subgenomic mRNA molecule produces the structural proteins necessary for capsid and envelope formation.
Other RNA viruses which have a plus (+) strand genomic RNA, such as the flaviviruses and picornaviruses, do not synthesize subgenomic RNAs during their life cycle. Rather, these RNA viruses contain a single open reading frame for translation and the viral proteins are produced by co- and post-translational cleavage of a polyprotein. Flaviviruses are a genus of the Togaviridae family of viruses and include such pathogenic species as St. Louis encephalitis, Japanese B encephalitis, Murray Valley encephalitis, West Nile, Dengue, and Yellow Fever. The Picornaviruses include the Poliovirus, Coxsackievirus, Echovirus, Enterovirus and Rhinovirus. The clinical detection of each of these viruses is also important for the diagnosis of disease and for research purposes.
Heretofore, the detection of RNA viruses in a specimen has not included the use of indicator cell lines capable of detecting RNA viruses that replicate through an RNA intermediate and only more costly and laborious techniques have been utilized. It would be desirable, therefore, to provide a means for detecting the presence of an RNA virus in a specimen that utilizes a genetically engineered cell line so as to provide a rapid, sensitive and quantifiable in vitro assay for RNA viruses.